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| − | }</style><header class="d-flex justify-content-center"><div class="container d-flex align-items-end justify-content-between"><div id="heading"><h1>Safety</h1></div><a href="https://unsplash.com/photos/lZ4xZZuk8iA" rel="noopener" target="_blank"><span data-placement="top" id="bg-attribution" title="Photo by Josh Withers on Unsplash"></span></a></div></header><main class="hello theme-yellow"><div class="container"><div class="row"><div class="sidebar"><div class="nav" id="contents"><ul></ul></div></div><div class="content"><article ><h1>Fructose Regulated Anti-Invertase Construct</h1><p> The biosensor is the crux of our genetic circuit and is composed of Frur, pFrub and the Anti-invertase gene. Frur is a transcription factor with an affinity for D-fructose. The Frur gene coding for the FruR protein prevents transcription of the regulated promoters. pFruB is the promoter region following FruR and is repressed by the FruR transcription factor, in the absence of D-Fructose.</p>< div class="image"> <img alt="Fructose regulated Anti-Invertase Construct in the absence of Fructose-1 -phosphate" src="https:/ /static.igem.org/mediawiki/2020/6/6d/T--BITSPilani-Goa_India--img--Design--AIwoFruc.png"/><p> Figure 1: Fructose regulated Anti-Invertase Construct in the absence of Fructose-1-phosphate</p></div><p>This prevents any further transcription, and no Anti-invertase protein is released. If D-Fructose is present in the cell, the FruR transcription factor will bind preferentially to it and thus be inactivated. This means that the repression of the related promoter pFruB will be released, enabling the transcription of the anti-invertase protein. Which can then act on invertase and stop sucrose loss.</p><h1>Killswitch</h1><p>It is very important for us to have a robust biosafety model for our project because it is associated with a widely consumed edible product, sugar. The most important parts of the killswitch construct are the FNR promoter, the toxin ccdB responsible for binding and impairing the functioning of bacterial Gyrase and it’s antitoxin ccda. The ccda is under the FNR promoter whereas the ccdb is under a commonly used T7 constitutive promoter. Both promoters are supplemented by conserved and strong Ribosomal Binding Sites (RBS). The FNR promoter is only active in an anaerobic environment and inactivates in an aerobic environment. It forms the crux of the construct because it controls the antitoxin expression.</ p>< p> The killswitch function can also be best explained with the help of two different cases. One where the bacterial cell is in an aerobic environment and one in an anaerobic environment. </ p>< h2> Anaerobic</ h2>< p> This Diagram portrays the behaviour of the killswitch genetic circuit inside the sugarcane stem which is an anaerobic environment.</ p><p> The ccdb toxin is under a constitutive promoter which constantly expresses the toxin inside the cell. Due to the nature of the surrounding, the FNR promoter will be active and hence ccda antitoxin will also be produced in the cell. Since both ccda and ccdb is present in the cell, they will make a toxin-antitoxin complex and be degraded. This way the bacteria is able to survive inside the sugarcane stem.</p>< h2> Aerobic</ h2>< p> This diagram portrays the behaviour of the killswitch genetic circuit outside the sugarcane stem which is an aerobic environment.< /p> <div class="image ">< img alt="Killswitch Genetic Circut outside sugarcane stem" src="https:/ /static.igem.org/mediawiki/2020/0/07/T--BITSPilani-Goa_India--img--Design--KSAero.png"/><p> Figure 2: Killswitch Genetic Circut outside sugarcane stem</ p></div><p> In this case, too, ccdb is expressed constitutively with the help of the T7 promoter. But due to the presence of oxygen, the FNR promoter is switched off. This leads to an increase in the levels of ccdb in the cell. Which in turn leads to a complete halt in cell division and death. This ensures that the bacteria will not be able to survive outside the sugarcane environment.< /p><div class=" image">< img alt="Anti-Invertase Modelled Protein Structure" src="https:/ /static. igem. org/mediawiki/2020/d/db/T--BITSPilani-Goa_India--img--Design--AI.gif"/><p>Figure 3: Anti-Invertase Modelled Protein Structure</p ></div></article><div class="d-flex justify-content-between my-5"><a class="button prev flex justify-content-end long" href="https://2020.igem.org/Team:BITSPilani-Goa_India/Description"><span aria-hidden="" class="circle"><span class="icon arrow"></span></span><span class="button-text">Description</span></a><a class="button next short" href="https://2020.igem.org/Team:BITSPilani-Goa_India/Model"><span aria-hidden="" class="circle"><span class="icon arrow"></span></span><span class="button-text">Model</span></a></div></div></div></div></main><footer><div id="footerNav"><div class="container"><div class="row justify-content-between"><div class="col-sm-6 col-md-4 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class="content"><article><p> Biosafety is the ethical obligation of a bioengineer. Throughout our project design we have tried to be as mindful as possible regarding identifying strategies for robust biosafety. Since our project is closely related to a consumable sugar, we have devised a three-tier fail-safe mechanism after taking into consideration the various use cases of our chassis.</p>< h1> Level 1: Kill Switch</ h1><p> We intend to introduce our strain of bacteria into the sugarcane matrix to synthesise anti-invertase. Considering the close proximity of the system to both agricultural environments and human consumables, it is indispensable that our bacteria include a lethality mechanism. We propose to use a modified type II ccdA-ccdB toxin-antitoxin system as our kill switch that is activated upon exposure to atmospheric concentrations of oxygen. A change in the oxygen concentration in the environment results in a sharp increase in the amount of the toxin protein being synthesised which disturbs the already established equilibrium between the toxin and antitoxin, effectively killing the cell, and thus preventing any contamination by stray microbes. More information about the design of the kill switch can be found < a href="https:/ /2020.igem.org/Team:BITSPilani-Goa_India/Safety/Design#KillSwitch"> here< /a> and a study of its dynamics <a href="https://2020. igem. org/ Team:BITSPilani-Goa_India/Safety/Model/Kill_Switch"> here< /a> .</ p>< h1> Level 2: Auxotropic Regulation</ h1><p> There are several amino acids that have been detected in sugarcane including, pipecolic acid and hydroxypipecolic acid. Increasing the supply of nitrogen increases markedly the amides asparagine and glutamine and less markedly the basic amino‐acids lysine, histidine, arginine and tryptophan. The neutral and acidic amino‐acids tend to increase and then decrease in level with increasing nitrogen supply. An auxotroph of tryptophan would</p>< h1> Level 3: Sugar Manufacturing Process</ h1>< blockquote><p> Need better quality image of the industy process</p></ blockquote><p> During the sugar manufacturing process the cane juice is heated at the evaporation station to temperatures greater than 100°C for upto eught hours. Furthermore, sulphur dioxide is passed through the solution at the clarifying stage along with a change in pH. We have designed experiments to mimic these industrial conditions on a lab scale. More details about the same can be found < a href=" #Experiments"> here</ a>. The bacteria would not be able to handle the stress of these conditions and hence the manufacturing process would act as a third failsafe.</p></article><div class="d-flex justify-content-between my-5"><a class="button prev flex justify-content-end long" href="https://2020.igem.org/Team:BITSPilani-Goa_India/Description"><span aria-hidden="" class="circle"><span class="icon arrow"></span></span><span class="button-text">Description</span></a><a class="button next short" href="https://2020.igem.org/Team:BITSPilani-Goa_India/Model"><span aria-hidden="" class="circle"><span class="icon arrow"></span></span><span class="button-text">Model</span></a></div></div></div></div></main><footer><div id="footerNav"><div class="container"><div class="row justify-content-between"><div class="col-sm-6 col-md-4 col-lg-3 py-2"><a class="footer-heading">Project</a><ul><li class="py-1"><a href="https://2020.igem.org/Team:BITSPilani-Goa_India/Description">Description</a></li><li class="py-1"><a 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